In this proposal, we investigate the mechanisms and implications of the regulation of the Notch signaling pathway by fringe. Notch signaling is of great biological and medical importance, because most animal tissues appear to utilize Notch signaling for at least some aspects of their development. The range of human diseases that have been associated with mutations in the Notch pathway thus far reflects the diversity of Notch functions in human biology: aberrant Notch signaling has been linked to leukemia, a congenital syndrome associated with stroke and dementia, a congenital syndrome associated with liver, cardiovascular, skeletal and other defects, and a congenital syndrome associated with axial skeletal defects. Fringe functions as a modulator of the Notch signaling pathway and encodes a novel glycosyltransferase that elongates 0-linked fucose. The projects outlined in this renewal application are directed towards understanding how Fringe-dependent carbohydrate modifications influence Notch activation and other cellular processes. To elucidate the mechanisms by which Fringe modulates Notch activation, we will identify the proteins, and the EGF modules within those proteins, that are the biologically relevant targets of Fringe by altering consensus sites for addition of 0-linked fucose within Notch, Serrate, and Delta, and assaying the activity of these mutant proteins in Drosophila. We will also analyze the role of distinct carbohydrate structures on Notch by analyzing the expression and in vivo requirements for a polypeptide fucosyltransferase and a B-galactoside sialyltransferase. In order to define the molecular mechanism by which Fringe modulates Notch signaling, we will establish an in vitro Notch-ligand binding assay. We will then use this assay to relate the influence of alterations in specific EGF domains and their carbohydrate structures on Notch activation in vivo to the physical interactions between Notch and its ligands. The positioning of Notch activation by Fringe separates the Drosophila wing into distinct dorsal and ventral compartments. We will test our hypothesis that mitoses are oriented parallel to the compartment border by directly visualizing mitotic spindles. We will then assess the role and regulation of oriented mitoses in the wing by analyzing the influence of members of the tissue polarity pathway and the Notch pathway on this process. These studies will advance our understanding of the regulation and functions of the Notch signaling pathway, and will provide the first detailed picture of how differential receptor glycosylation can influence receptor-ligand interactions.